Therapeutic Efficacy Of Lentiviral Vector Mediated Insulin Like Growth Factor-1Gene-modified Muscle-derived Stem Cells In Rats With Female Stress Urinary Incontinence

Objective To explore the treatment effectiveness of lentiviral vector mediated insulinlike growth factor-1(IGF-1) gene-modified muscle-derived stem cells (MDSCs) forrat models of female stress urinary incontinence and its related meehanism.Methods1. Separation, purification, identification and differentiation of the primaryrat MDSCs in vitro. After mechanical and enzymatic digestion method, MDSCs wereisolated and cultivated by modified preplating process. By inverted phase contrastmicroscope, we observed the growth, morphological changes and differentiation ofMDSCs, whose surface antigens were identified by flow cytometry,immunofluorescence and immunohistochemical method respectively. The myotubeswere tesed by the expression of MyHC protein via immunofluorescence method.2.By infusion technology In vitro, IGF-1recombinant lentiviral vector (pGC-FU)carrying green fluorescent protein (GFP) was constructed and then was identified bypolymerase chain reaction (PCR), restriction enzyme digestion and sequencingidentified. After IGF-1recombinant lentiviral vector was transfected into293T cells.The lentiviral titer was detected by real-time fluorescence quantitative PCR (RTQ-PCR). The expression of GFP was observed by fluorescence microscopy andwestern blot. By the expression of fluorescence and the secretion lever of IGF-1protein, the optimal MOI (MOI) was confirmed.3. MDSCs were infected byLenti-IGF-1or Lenti-GFP, which was named as IGF-1/MDSCs or GFP/MDSCs,respectively. After they were cultured90%fusion, hydrogen peroxide (H₂O₂) wasused to imitation the oxidative stress microenvironment in vitro. Apoptosis weredetected by DNA ladder and FCM. The expressions of P-AKT, NFκB, Bcl-2, Bcl-xl,caspases-3and PARP were determined by Western blot.4. Bilateral pudendalnerve-transected (PNT) in female rats were applies as stress urinary incontinence (SUI) models.60grown-up rat were divide equally stochastically into thesham-operation group (S group), the control group (PBS group), the GFP/MDSCsgroup and the IGF-1/MDSCs group.1×106MDSCs/rat were injected into theproximal uretha.The urodynamics was applied to assessed whether the modelssucceeded or not and the uretha closed function after injection. The surviving cellswere tracked by laser scanning confocal microscope (LSCM). Their differentiationinto myotubes-like cells were evaluated by immunofluorescent stainning. Urethablood capillary were detected by the expression of Factor VIII-related Antigen. TheHE and the Masson dyeing were used to assessed the pathological changes afterinjection. The expresses of insulin like growth factor-1(IGF-1), vascular endothelialcell growth factor (VEGF) in uretha zone were detected by immunity histochemistryand Western blot.Results1. MDSCs were isolated and cultured with low adhesion, small round,refraction. They may differentiate into the myotubes. Their phenotype was identifiedas Sca-1(+), CD34(+), CD45(-) and desmin (+), which was in line with thecharacteristics of MDSCs.2. By PCR, restriction digestion and sequencing, IGF-1＆eGFP recombinant lentiviral vector was correct. The titer of the packaged lentiviruswas about2×108TU/ml and the optimum MOI is30.3. Though the DNA ladder, FCMand the expressions of caspase-3, PARP, P-AKT, NFκB, Bcl-2and Bcl-xl by Westernblot, we found that H₂O₂could induce MDSCs endogenous caspaseenzyme-dependent apoptosis pathway and IGF-1can antagonize the effect of H₂O₂byPI3K/AKT signal path.4. The construction of stress urinary incontinence rat modelswere successful. Cell viability was higher in IGF-1/MDSCs group than inGFP/MDSCs group after one week cells injection. The expressions of MyHC weremore in IGF-1/MDSCs group than in GFP/MDSCs group four weeks after cellinjection. The ALPP and MBC were more higher in IGF-1/MDSCs and GFP/MDSCsgroup at every time point (p<0.01). The microvessel density was higher in IGF-1/MDSCs group than in other groups four weeks after cell injection (p<0.01). TheMasson dyeing showed that an more muscle/collagen ratio was observed in IGF-1/MDSCs group((p<0.05). The expressios of IGF-1, VEGF in the uretha zonewere higher in GFP/MDSCs group within two weeks after cell transplantation than inPBS group(p<0.05). Expression of these growth factors in IGF-1/MDSCs were higherthan other groups in every time point (p<0.05).Conclusions MDSCs can be isolated and purified successfully from Wistar rats.IGF-1gene-modified lentiviral vector can be constructed safely and transfectedefficiently into rat MDSCs. IGF-1gene modification enhanced survival of MDSCsunder oxidative stress microenvironment, and transplantation with IGF-1engineeredMDSCs may enhance substantial functional recovery after transplanted cells on SUIrat models.